Differential ability of heparan sulfate proteoglycans to assemble the fibroblast growth factor receptor complex in situ.

Fibroblast growth factors (FGFs) require heparan sulfate proteoglycans (HSPGs) as cofactors for signaling. The heparan sulfate chains (HS) mediate stable high affinity binding of FGFs to their receptor tyrosine kinases (FR) and may specifically regulate FGF activity. A novel in situ binding assay was developed to examine the ability of HSPGs to promote FGF/FR binding using a soluble FR fusion construct (FR1-AP). This fusion protein probe forms a dimer in solution, simulating the dimerization or oligomerization that is thought to occur at the cell surface physiologically. In frozen sections of human skin, FGF-2 binds to keratinocytes and basement membranes of epidermis and dermal blood vessels. In contrast, in skin preincubated with FGF-2, FR1-AP binds avidly to FGF-2 immobilized on keratinocyte cell surfaces, but fails to bind to basement membranes at the dermo-epidermal junction or dermal microvessels despite the fact that these structures bind large amounts of FGF-2. Apparently, basement membrane and cell surface HSPGs differ in their ability to mediate the assembly of a FGF/FR signaling complex presumably due to structural differences of the heparan sulfate chains.     

Heparan sulfate-modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through the receptor tyrosine kinase c-Met

CD44 has been implicated in tumor progression and metastasis, but the mechanism(s) involved is as yet poorly understood. Recent studies have shown that CD44 isoforms containing the alternatively spliced exon v3 carry heparan sulfate side chains and are able to bind heparin-binding growth factors. In the present study, we have explored the possibility of a physical and functional interaction between CD44 and hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the receptor tyrosine kinase c-Met. The HGF/SF-c-Met pathway mediates cell growth and motility and has been implicated in tumor invasion and metastasis. We demonstrate that a CD44v3 splice variant efficiently binds HGF/SF via its heparan sulfate side chain. To address the functional relevance of this interaction, Namalwa Burkitt's lymphoma cells were stably co-transfected with c-Met and either CD44v3 or the isoform CD44s, which lacks heparan sulfate. We show that, as compared with CD44s, CD44v3 promotes: (i) HGF/SF-induced phosphorylation of c-Met, (ii) phosphorylation of several downstream proteins, and (iii) activation of the MAP kinases ERK1 and -2. By heparitinase treatment and the use of a mutant HGF/SF with greatly decreased affinity for heparan sulfate, we show that the enhancement of c-Met signal transduction induced by CD44v3 was critically dependent on heparan sulfate moieties. Our results identify heparan sulfate-modified CD44 (CD44-HS) as a functional co-receptor for HGF/SF which promotes signaling through the receptor tyrosine kinase c-Met, presumably by concentrating and presenting HGF/SF. As both CD44-HS and c-Met are overexpressed on several types of tumors, we propose that the observed functional collaboration might be instrumental in promoting tumor growth and metastasis.     

Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development.

The Drosophila sugarless and sulfateless genes encode enzymes required for the biosynthesis of heparan sulfate glycosaminoglycans. Biochemical studies have shown that heparan sulfate glycosaminoglycans are involved in signaling by fibroblast growth factor receptors, but evidence for such a requirement in an intact organism has not been available. We now demonstrate that sugarless and sulfateless mutant embryos have phenotypes similar to those lacking the functions of two Drosophila fibroblast growth factor receptors, Heartless and Breathless. Moreover, both Heartless- and Breathless-dependent MAPK activation is significantly reduced in embryos which fail to synthesize heparan sulfate glycosaminoglycans. Consistent with an involvement of Sulfateless and Sugarless in fibroblast growth factor receptor signaling, a constitutively activated form of Heartless partially rescues sugarless and sulfateless mutants, and dosage-sensitive interactions occur between heartless and the heparan sulfate glycosaminoglycan biosynthetic enzyme genes. We also find that overexpression of Branchless, the Breathless ligand, can partially overcome the requirement of Sugarless and Sulfateless for Breathless activity. These results provide the first genetic evidence that heparan sulfate glycosaminoglycans are essential for fibroblast growth factor receptor signaling in a well defined developmental context, and support a model in which heparan sulfate glycosaminoglycans facilitate fibroblast growth factor ligand and/or ligand-receptor oligomerization.     

Enhancement of fibroblast growth factor (FGF) activity by an FGF-binding protein

Fibroblast growth factor-binding protein (FGF-BP) 1 is a secreted protein that can bind fibroblast growth factors (FGFs) 1 and 2. These FGFs are typically stored on heparan sulfate proteoglycans in the extracellular matrix in an inactive form, and it has been proposed that FGF-BP1 functions as a chaperone molecule that can mobilize locally stored FGF and present the growth factor to its tyrosine kinase receptor. FGF-BP1 is up-regulated in squamous cell, colon, and breast cancers and can act as an angiogenic switch during malignant progression of epithelial cells. For the present studies, we focused on FGF-1 and -2 and investigated interactions with recombinant human FGF-BP1 protein as well as effects on signal transduction, cell proliferation, and angiogenesis. We show that recombinant FGF-BP1 specifically binds FGF-2 and that this binding is inhibited by FGF-1, heparan sulfate, and heparinoids. Furthermore, FGF-BP1 enhances FGF-1- and FGF-2-dependent proliferation of NIH-3T3 fibroblasts and FGF-2-induced extracellular signal-regulated kinase 2 phosphorylation. Finally, in the chicken chorioallantoic membrane angiogenesis assay, FGF-BP1 synergizes with exogenously added FGF-2. We conclude that FGF-BP1 binds directly to FGF-1 and FGF-2 and positively modulates the biological activities of these growth factors.     

The interactions of hepatocyte growth factor/scatter factor and its NK1 and NK2 variants with glycosaminoglycans using a modified gel mobility shift assay. Elucidation of the minimal size of binding and activatory oligosaccharides

Full-length hepatocyte growth factor/scatter factor interacts with both heparan and dermatan sulfates and is critically dependent upon them as cofactors for activation of the tyrosine kinase receptor Met. Two C-terminally truncated variants (NK1 and NK2) of this growth factor also occur naturally. Their glycosaminoglycan binding properties are not clear. We have undertaken a comparative study of the heparan/dermatan sulfate binding characteristics of all three proteins. This has entailed the development of a modified gel mobility shift assay, utilizing fluorescence end-tagged oligosaccharides, that is also widely applicable to the analysis of many glycosaminoglycan-protein interactions. Using this we have shown that all three hepatocyte growth factor/scatter factor variants share identical heparan/dermatan sulfate binding properties and that both glycosaminoglycans occupy the same binding site. The minimal size of the oligosaccharide that binds with high affinity in all cases is a tetrasaccharide from heparan sulfate but a hexasaccharide from dermatan sulfate. These findings demonstrate that functional glycosaminoglycan binding is restricted to a binding site situated solely within the small N-terminal domain. The same minimal size fractions are also able to promote hepatocyte growth factor/scatter factor-mediated activation of Met and consequent downstream signaling in the glycosaminoglycan-deficient Chinese hamster ovary pgsA-745 cells. A covalent complex of heparan sulfate tetrasaccharide with monovalent growth factor is also active. The binding and activity of tetrasaccharides put constraints upon the possible interactions and molecular geometry within the ternary signaling complex.     

Potentiation and inhibition of bFGF binding by heparin: a model for regulation of cellular response

Basic fibroblast growth factor (bFGF) binds to cell surface tyrosine kinase receptor proteins and to heparan sulfate proteoglycans. The interaction of bFGF with heparan sulfate on the cell surface has been demonstrated to impact receptor binding and biological activity. bFGF receptor binding affinity is reduced on cells that do not express heparan sulfate. The addition of soluble heparin or heparan sulfate has been demonstrated to rescue the bFGF receptor binding affinity on heparan sulfate deficient cells yet has also been shown to inhibit binding under some conditions. While the chemical requirements of the heparin-bFGF-receptor interactions have been studied in detail, the possibility that heparin enhances bFGF binding in part by physically associating with the cell surface has not been fully evaluated. In the study presented here, we have investigated the possibility that heparin binding to the cell surface might play a role in modulating bFGF receptor binding and activity. Balb/c3T3 cells were treated with various concentrations of sodium chlorate, so as to express a range of endogenous heparan sulfate sites, and [(125)I]bFGF binding was assessed in the presence of a range of heparin concentrations. Low concentrations of heparin (0.1-30 nM) enhanced bFGF receptor binding to an extent that was inversely proportional to the amount of endogenous heparan sulfate sites present. At high concentrations (10 microM), heparin inhibited bFGF receptor binding in cells under all conditions. The ability of heparin to stimulate and inhibit bFGF-receptor binding correlated with altered bFGF-stimulated tyrosine kinase activity and cell proliferation. Under control and chlorate-treated conditions, [(125) I]heparin was observed to bind with a high affinity to a large number of binding sites on the cells (K(d) = 57 and 50 nM with 3.5 x 10(6) and 3.6 x 10(6) sites/cell for control and chlorate-treated cells, respectively). A mathematical model of this process revealed that the dual functions of heparin in bFGF binding were accurately represented by heparin cell binding-mediated stimulation and soluble heparin-mediated inhibition of bFGF receptor binding.     

Heparin can activate a receptor tyrosine kinase.

Heparin, a densely sulfated glycosaminoglycan produced by mast cells, is best known for its inhibitory effects on the blood coagulation system. Heparin or heparan sulfate proteoglycans are also essential cofactors for the interaction of fibroblast growth factors (FGFs) with their receptor tyrosine kinases (FGFRs). Here we show that heparin is a growth factor-independent activating ligand for FGFR-4. Heparin stimulates FGFR-4 autophosphorylation on transfected myoblasts, fibroblasts and lymphoid cells, and is most potent on cells lacking surface heparan proteoglycan. Two functional analogs of heparin, fucoidan and dextran sulfate, are also activators of FGFR-4, while neither heparin nor its analogs can stimulate FGFR-1 in the absence of FGF. A mutation in the FGFR-4 ectodomain which impairs receptor activation by FGFs does not interfere with activation by heparin, demonstrating that receptor domains required for heparin or FGF activation are not identical. Heparin activation of FGFR-4 or of a chimeric receptor bearing FGFR-4 ectodomain and FGFR-1 cytodomain triggers downstream tyrosine phosphorylation of several signaling proteins, and induces proliferation of cells bearing the chimeric receptor. Consistent with these findings, a soluble FGFR-4 ectodomain has strong FGF-independent affinity for immobilized heparin resin, while soluble FGFR-1 requires FGF for stable heparin interaction. Heparin activation of FGFR-4 is the first example of a mammalian polysaccharide serving as a signaling ligand.     

Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

Cell surface heparan sulfate (HS) not only binds several major classes of growth factors but also sometimes potentiates their activities—an effect usually termed “coreception.” A view that coreception is due to the stabilization of growth factor–receptor interactions has emerged primarily from studies of the fibroblast growth factors (FGFs). Recent in vivo studies have strongly suggested that HS also plays an important role in regulating signaling by the bone morphogenetic proteins (BMPs). Here, we provide evidence that the mechanism of coreception for BMPs is markedly different from that established for FGFs. First, we demonstrate a direct, stimulatory role for cell surface HS in the immediate signaling activities of BMP2 and BMP4, and we provide evidence that HS–BMP interactions are required for this effect. Next, using several independent assays of ligand binding and receptor assembly, including coimmunoprecipitation, cross-linking, and fluorescence fluctuation microscopy, we show that HS does not affect BMP binding to type I receptor subunits but instead enhances the subsequent recruitment of type II receptor subunits to BMP-type I receptor complexes. This suggests a view of HS as a catalyst of the formation of signaling complexes, rather than as a stabilizer of growth factor binding.     

FGFR3 Transmembrane Domain Interactions Persist in the Presence of Its Extracellular Domain

Isolated receptor tyrosine kinase transmembrane (TM) domains have been shown to form sequence-specific dimers in membranes. Yet, it is not clear whether studies of isolated TM domains yield knowledge that is relevant to full-length receptors or whether the large glycosylated extracellular domains alter the interactions between the TM helices. Here, we address this question by quantifying the effect of the pathogenic A391E TM domain mutation on the stability of the fibroblast growth factor receptor 3 dimer in the presence of the extracellular domain and comparing these results to the case of the isolated TM fibroblast growth factor receptor 3 domains. We perform the measurements in plasma membrane-derived vesicles using a Förster-resonance-energy-transfer-based method. The effect of the mutation on dimer stability in both cases is the same (∼−1.5 kcal/mol), suggesting that the interactions observed in simple TM-peptide model systems are relevant in a biological context.     

Molecular networks in FGF signaling: flotillin-1 and cbl-associated protein compete for the binding to fibroblast growth factor receptor substrate 2

Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway.     

The protein core of the proteoglycan perlecan binds specifically to fibroblast growth factor-7

Perlecan is a multifaceted heparan sulfate proteoglycan that is expressed not only as an intrinsic constituent of basement membranes but also as a cell-surface and pericellular proteoglycan. Perlecan functions as a ligand reservoir for various growth factors that become stabilized against misfolding or proteolysis and acts as a co-receptor for basic fibroblast growth factor by augmenting high affinity binding and receptor activation. These biological properties are mediated by the heparan sulfate moiety. Rather little is known about the protein core's mediation of functions. We have recently discovered that fibroblast growth factor-7 (FGF7) binds to perlecan protein core and that exogenous perlecan efficiently reconstitutes FGF7 mitogenic activity in perlecan-deficient cells. In this report we examined the specific binding of FGF7 to various domains and subdomains of perlecan protein core. Using several experimental approaches including overlay protein assays, radioligand binding experiments, and the yeast two-hybrid system, we demonstrate that FGF7 binds specifically to the N-terminal half of domain III and to a lesser extent to domain V, with affinity constants in the range of 60 nM. Thus, perlecan protein core should be considered a novel biological ligand for FGF7, an interaction that could influence cancer growth and tissue remodeling.     

Binding of heparin/heparan sulfate to fibroblast growth factor receptor 4

Fibroblast growth factors (FGFs) are heparin-binding polypeptides that affect the growth, differentiation, and migration of many cell types. FGFs signal by binding and activating cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase domains. The signaling involves ligand-induced receptor dimerization and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both FGFs and FGFRs and enhance FGF signaling by mediating complex formation between the growth factor and receptor components. Whereas the heparin/heparan sulfate structures involved in FGF binding have been studied in some detail, little information has been available on saccharide structures mediating binding to FGFRs. We have performed structural characterization of heparin/heparan sulfate oligosaccharides with affinity toward FGFR4. The binding of heparin oligosaccharides to FGFR4 increased with increasing fragment length, the minimal binding domains being contained within eight monosaccharide units. The FGFR4-binding saccharide domains contained both 2-O-sulfated iduronic acid and 6-O-sulfated N-sulfoglucosamine residues, as shown by experiments with selectively desulfated heparin, compositional disaccharide analysis, and a novel exoenzyme-based sequence analysis of heparan sulfate oligosaccharides. Structurally distinct heparan sulfate octasaccharides differed in binding to FGFR4. Sequence analysis suggested that the affinity of the interaction depended on the number of 6-O-sulfate groups but not on their precise location.     

The single transmembrane domains of ErbB receptors self-associate in cell membranes.

Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) alpha-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM alpha-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers. In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo. Using an approach for analyzing TM domain interactions in Escherichia coli cell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM equivalent to ErbB2-TM > ErbB3-TM. A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer. We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor. Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization. Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups.     

The Acidic Domain and First Immunoglobulin-Like Loop of Fibroblast Growth Factor Receptor 2 Modulate Downstream Signaling through Glycosaminoglycan Modification

Fibroblast growth factor receptors (FGFRs) are membrane-spanning tyrosine kinases that have been implicated in a variety of biological processes including mitogenesis, cell migration, development, and differentiation. We identified a unique isoform of FGFR2 expressed as a diffuse band with an unusually large molecular mass. This receptor is modified by glycosaminoglycan at a Ser residue located immediately N terminal to the acidic box, a stretch of acidic amino acids. The acidic box and the glycosaminoglycan modification site are encoded by an alternative exon of the FGFR2 gene. The acidic box appears to play an important role in glycosaminoglycan modification, and the presence of this domain is required for modification by heparan sulfate glycosaminoglycan. Moreover, the presence of the first immunoglobulin-like domain encoded by another alternative exon abrogated the modification. The high-affinity receptor with heparan sulfate modification enhanced receptor autophosphorylation, substrate phosphorylation, and ternary complex factor-independent gene expression. It also sustained mitogen-activated protein kinase activity and increased eventual DNA synthesis, a long-term response to fibroblast growth factor stimulation, at physiological ligand concentrations. We propose a novel regulation mechanism of FGFR2 signal transduction through glycosaminoglycan modification.     

Binding of interferon-gamma to heparan sulfate is restricted to the heparin-like domains and involves carboxylic--but not N-sulfated--groups.

Interferon-gamma binds to the glycosaminoglycan part of basement membrane proteoglycan. To obtain a greater insight into this interaction, different glycosaminoglycans and their subfractions were used in various binding assays. High affinity binding occurs with heparin and heparan sulfate only, the latter being the predominant basement membrane glycosaminoglycan. Furthermore, using heparan sulfate and heparin treated with heparinases I and III, we have shown that the interferon-gamma binding sites are localized on the N-sulfated glucosamine rich domains of the molecule. Interestingly, interferon-gamma and fibroblast growth factor compete for the same binding domain on heparan sulfate, although they are unrelated proteins. This last point is discussed in the light of the conformational flexibility of the glycosaminoglycan molecules.     

Common binding sites for beta-amyloid fibrils and fibroblast growth factor-2 in heparan sulfate from human cerebral cortex.

Heparan sulfate found in the cerebral plaques of Alzheimer's disease binds to beta-amyloid (Abeta) fibrils. This interaction has been proposed to enhance fibril deposition and mediate Abeta-induced glia activation and neurotoxicity. On the other hand, heparan sulfate augments signaling of fibroblast growth factor-2 (FGF-2), a neuroprotective factor that antagonizes the neurotoxic effects of Abeta. We defined structures in heparan sulfate from human cerebral cortex that bind Abeta fibrils. The minimal binding site is found in N-sulfated hexasaccharide domains and contains critical 2-O-sulfated iduronic acid residues. By contrast, binding of Abeta monomers requires, in addition, 6-O-sulfate groups on glucosamine residues. The binding specificity of fibrillar Abeta is shared by FGF-2, and we here show that cerebral heparan sulfate domains selected for binding to Abeta-(1-40) fibrils bind also to FGF-2. These data suggest that neurotoxic and neuroprotective signals may converge by competing for the same binding sites on the heparan sulfate chain.     

Heparan sulfate primed on β-D-xylosides restores binding of basic fibroblast growth factor

Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF). Mutant Chinese hamster ovary cells (pgsA-745) deficient in xylosyltransferase are unable to initiate glycosaminoglycan synthesis and hence can not bind bFGF to low- and high-affinity cell surface receptors. Exposure of pgsA-745 cells to β-D-xylopyranosides containing hydrophobic aglycones resulted in restoration of bFGF binding in a manner similar to that induced by soluble heparin or by heparan sulfate (HS) normally associated with cell sulfate. Restoration of bFGF binding correlated with the ability of the β-D-xylosides to prime the synthesis of heparan sulfate. Thus, both heparan sulfate synthesis and bFGF receptor binding were induced by low concentrations (10–30 μM) of estradiol-β-D-xyloside and naphthyl-β-D-xyloside, but not by cis/trans-decahydro-2-naphthyl-β-D-xyloside, which at low concentration primes mainly chondroitin sulfate. The obligatory involvement of xyloside-primed heparan sulfate in restoration of bFGF-receptor binding was also demonstrated by its sensitivity to heparinase treatment and by the lack of restoration activity in CHO cell mutants that lack enzymatic activities required to form the repeating disaccharide unit characteristic of heparan sulfate. Xyloside-primed heparan sulfate binds to the cell surface. Restoration of bFGF receptor binding was induced by both soluble and cell bound xyloside-primed heparan sulfate and was abolished in cells that were exposed to 0.5–1.0 M NaCl prior to the bFGF binding reaction. These results indicate that heparan sulfate chains produced on xyloside primers behave like heparan sulfate chains attached to cellular core proteins in terms of affinity for bFGF and ability to function as low-affinity sites in a dual receptor mechanism characteristic of bFGF and other heparin-binding growth promoting factors.     

Binding of two growth factor families to separate domains of the proteoglycan betaglycan.

Cell surface proteoglycans help present some polypeptide growth factors such as basic fibroblast growth factor (bFGF) to their receptors and may act as reservoirs for others such as transforming growth factor-beta (TGF-beta). Betaglycan, a cell surface heparan sulfate/chondroitin sulfate proteoglycan that binds TGF-beta via its core protein, is shown here to bind bFGF via its heparan sulfate chains. We investigated the potential for regulation of betaglycan by its ligands in osteoblasts, a system in which bFGF and TGF-beta have complementary effects. We report here that the apparent molecular mass of betaglycan from an osteoblast-enriched primary culture of fetal rat calvaria is decreased in response to bFGF, as detected by an increased electrophoretic migration of betaglycan. The betaglycan forms expressed in bFGF-treated osteoblasts have a reduced content of heparan sulfate GAGs, without detectable changes in the content of chondroitin sulfate GAGs or the size of the core protein. bFGF did not affect the overall population of cell-surface-associated proteins identified by sulfate labeling, which contained primarily heparan sulfate, and had only small effects on the major secreted proteoglycans, which were, by contrast, chondroitin sulfate proteoglycans. The effect of bFGF on betaglycan is therefore a selective one. These results suggest that cells can interact with members of the TGF-beta and FGF families through separate domains of the same membrane proteoglycan, and can selectively regulate the bFGF-binding carbohydrate chains of this proteoglycan in response to bFGF.     

Requirement for anticoagulant heparan sulfate in the fibroblast growth factor receptor complex.

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the fibroblast growth factor (FGF) receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation between self-associated FGFR and determines specificity for and mediates binding of activating FGF. Here we show that only the fraction of commercial heparin or rat liver heparan sulfate which binds to immobilized antithrombin formed an FGF-binding binary complex with the ectodomain of the FGFR kinase. Conversely, only the fraction of heparin that binds to immobilized FGFR inhibited Factor Xa in the presence of antithrombin. Only the antithrombin-bound fraction of heparin competed with (3)H-heparin bound to FGFR in absence of FGF, whereas both antithrombin-bound and unretained fractions competed with radiolabeled heparin bound independently to FGF-1 and FGF-2. The antithrombin-bound fraction of heparin was required to support the heparin-dependent stimulation of DNA synthesis of endothelial cells by FGF-1. The requirement for divalent cations and the antithrombin-binding motif distinguish the role of heparan sulfate as an integral subunit of the FGFR complex from the wider range of effects of heparan sulfates and homologues on FGF signaling through FGFR-independent interactions with FGF.     

Cell- and receptor isotype-specific phosphorylation of SNT1 by fibroblast growth factor receptor tyrosine kinases

A partnership between the ectodomain of the fibroblast growth factor receptor (FGFR) isotypes and the chains of pericellular matrix heparan sulfate determines the fibroblast growth factor (FGF) and cell-type specificitives of the FGFR signaling complex. The contribution of the FGFR intracellular tyrosine kinase domains to the specificity of FGFR signaling is unclear. This report shows that the quantity and quality of phosphorylation of the FGFR kinase substrate SNT1 (also called FGFR substrate 2, FRS2) is both FGFR isotype and cell-type specific in prostate tumor epithelial cells at different stages of malignancy. Epithelial cell-resident FGFR2 that promotes homeostasis yields a low level of phosphorylated 65-kDa SNT1. Phosphorylation by ectopic FGFR1 that promotes malignancy was much more intense and yielded a phosphorylated 85-kDa SNT1. The amount of the 85-kDa SNT1 increased by 20-fold during proliferative aging of FGFR1-expressing cell populations that is required for FGFR1-stimulated mitogenesis and the malignant phenotype. In addition, the receptor-specific differential phosphorylation of SNT1 by FGFR isotypes, both of which are normally anchored to the cell membrane, occurred only in intact cells. Therefore, similar to kinase subunits within the heparan sulfate-FGFR complex, cell membrane and cytoskeletal context likely determine FGFR isotype- and cell-type-specific conformational relationships between FGFR kinases and external substrates. This determines the quantity and quality of SNT1 phosphorylation and differential signaling.